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original article

& 2006 International Society of Nephrology

Efficacy of local dipyridamole therapy in a porcine model of arteriovenous graft stenosis T Kuji1, T Masaki1, K Goteti2, L Li1, S Zhuplatov1, CM Terry1, W Zhu2, JK Leypoldt1,3,4, R Rathi5, DK Blumenthal6, SE Kern2,3 and AK Cheung1,7 1

Department of Medicine, University of Utah, Salt Lake City, Utah, USA; 2Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah, USA; 3Department of Bioengineering, University of Utah, Salt Lake City, Utah, USA; 4Research Service, Veterans Affairs Salt Lake City Healthcare System, Salt Lake City, Utah, USA; 5MacroMed Inc., Sandy, Utah, USA; 6Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, USA and 7Medical Service, Veterans Affairs Salt Lake City Healthcare System, Salt Lake City, Utah, USA

Perivascular delivery of antiproliferative drugs has been proposed as an approach to prevent neointimal hyperplasia associated with hemodialysis polytetrafluoroethylene (PTFE) grafts. We examined this approach to deliver dipyridamole in a porcine graft model. PTFE grafts were implanted between the carotid artery and external jugular vein bilaterally in pigs. During the surgery or 1 week post-graft placement, dipyridamole (0.26–52 mg) alone or incorporated in microspheres was mixed with an injectable polymeric gel and applied to the graft-arterial and graft-venous anastomoses on one side, whereas the contralateral control graft received no treatment. Three or four weeks after operation, the grafts and adjacent vessels were explanted en bloc and crosssections of the anastomoses were examined histologically. The degree of neointimal hyperplasia was quantified by planimetry. In separate experiments, dipyridamole was extracted from the explanted tissues and assayed by spectrofluorometry. The normalized median hyperplasia areas of the treated and control graft-venous anastomoses were 0.45 (25th–75th percentile, 0.30–0.86) and 0.24 (0.21–0.30), respectively (N ¼ 7; P ¼ 0.08). The median hyperplasia areas of the treated and control graft-arterial anastomoses were 0.12 (0.07–0.39) and 0.11 (0.09–0.13), respectively (N ¼ 7; P ¼ 0.31). The dipyridamole levels in the vascular walls around the anastomoses were at or above the in vitro inhibitory concentrations for approximately 3 weeks. These results suggest that the local perivascular sustained delivery of dipyridamole, even at high dosages, was ineffective in inhibiting neointimal hyperplasia associated with PTFE grafts in a porcine model. Kidney International (2006) 69, 2179–2185. doi:10.1038/sj.ki.5000383; published online 3 May 2006 KEYWORDS: dipyridamole; drug delivery; PTFE graft; hemodialysis vascular access; neointimal hyperplasia Correspondence: AK Cheung, Dialysis Program, University of Utah, 85 North Medical Drive East, Salt Lake City, Utah 84112, USA. E-mail: [email protected] Received 16 December 2005; revised 18 January 2006; accepted 31 January 2006; published online 3 May 2006 Kidney International (2006) 69, 2179–2185

Stenosis followed by thrombosis is the major cause of failure of polytetrafluoroethylene (PTFE) grafts for chronic hemodialysis. Stenosis occurs most commonly at the graft-venous anastomosis, although the graft-arterial anastomosis is also affected.1 Histologically, the lesions consist mostly of myointimal hyperplasia, which is composed of proliferating vascular smooth muscle cells, extracellular matrix, and neo-microvasculature.2 Therefore, the inhibition of smooth muscle cell proliferation and migration would appear to be a reasonable approach to prevent graft stenosis. Although the pathogenesis of neointimal hyperplasia associated with dialysis grafts has not been fully elucidated, a number of contributing factors are probably operative on a chronic basis, beyond the initial surgical trauma. These include the mismatch of vessel sizes and compliance, localized flow disturbances, and the release of growth factors from the vascular wall and adhering platelets in response to dialysis needle puncture. Therefore, chronic rather than acute delivery of antiproliferative agents is likely to be necessary to prevent stenosis on a long-term basis. Dipyridamole is an antiproliferative drug with a primary mechanism of action thought to derive from inhibiting phosphodiesterases and subsequently increasing intracellular cyclic nucleotide levels.3 One clinical study has suggested that daily oral dipyridamole with or without aspirin decreased the incidence of graft occlusion in dialysis patients, although the inhibition of neointimal hyperplasia was not documented.4 In addition, the number of patients studied was small (o30 in each group). A large multicenter trial sponsored by the National Institutes of Health examining the efficacy of the oral combination of dipyridamole and aspirin in maintaining dialysis PTFE graft patency is ongoing.5 Because of the focal, superficial, and accessible nature of the hyperplastic lesion associated with dialysis grafts, direct application of an antiproliferative agent that produces high local concentrations and minimal systemic exposure is an attractive therapeutic strategy. In fact, systemic administration of dipyridamole has been found to be ineffective in preventing the development of neointimal hyperplasia 2179

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following interposition venous grafts to the femoral artery in the dog6 and interposition PTFE grafts in the aorta in the goat.7 In contrast, continuous infusion of dipyridamole into the adventitia for more than 2 weeks was successful in partially inhibiting intimal thickening after balloon injury in the carotid or femoral artery of the rabbit.8 This latter study suggests that the continuous local perivascular delivery of dipyridamole may be more efficacious than systemic administration. Our recent study has suggested that the local sustained delivery of the antiproliferative drug paclitaxel using an injectable polymeric gel (ReGels; see details in the Materials and Methods section) applied perivascularly was effective in inhibiting the neointimal hyperplasia at the graft anastomoses in a canine model.9 These promising data, in conjunction with the demonstration of greater potency of dipyridamole in inhibiting the proliferation of cultured vascular smooth muscle cells compared to paclitaxel,10 have led to the present study. We report herein the results of perivascular sustained delivery of dipyridamole in a porcine model of PTFE graft stenosis. RESULTS Establishment of porcine PTFE model of stenosis

Eleven pigs with bilateral graft placement without drug treatment were used to study the time-dependent progression of neointimal hyperplasia. Two of these animals died within 24 h after surgery from complications of anesthesia. The remaining nine animals were killed at 7 (n ¼ 1), 14 (n ¼ 2), 21 (n ¼ 2), or 28 days (n ¼ 4), respectively. No neointimal hyperplasia was found within 7 days of graft placement, whereas progressive hyperplasia was found after 14 days at the graft-venous (Figure 1) and graft-arterial anastomoses (not shown).

T Kuji et al.: Local dipyridamole in porcine graft stenosis

of the contralateral controls was 0.11 (0.09–0.13; N ¼ 7; P ¼ 0.31; Figure 3b). Local effects of drug depot

Local perivascular delivery of dipyridamole did not result in any overt problems at the site of graft implantation. There was no obvious bleeding from the surgical wound or delayed wound healing. No inflammatory reactions were visible on or through the skin. In four animals, however, histological examination showed the drug depot to be encapsulated. Figure 4 shows the hematoxylin and eosin (H&E) staining of a cross-section of the graft-venous anastomosis from an animal that received 52 mg of dipyridamole in microspheres/ ReGel intraoperatively and was killed 28 days later. The section clearly shows that the depot remained at the site of deposition near the anastomosis contiguous with the blood vessel. Despite the conspicuous presence of the drug depot, neointimal hyperplasia was readily seen in the adjacent lumen. Closer examination under high magnification shows that the depot was surrounded by a septated fibrous capsule (Figure 4, inset), which was confirmed by trichrome staining. Occasional giant cells were seen inside the capsule. The encapsulation was also found on histological examination in three other pigs treated with dipyridamole delivered by microspheres/ReGel. Target tissue concentrations of dipyridamole

High concentrations of dipyridamole were found in the arterial and venous wall at the anastomoses (Figure 5), indicating that the drug released from microspheres/ReGel readily diffused into the target sites. The mean concentrations remained above 100 mg/ml for at least 8 days and decreased to approximately 10 mg/ml by 21 days at both arterial and venous anastomoses.

Assessment of neointimal hyperplasia

Nine pigs were used to examine the efficacy of local dipyridamole treatment. In two of the nine animals, technical problems prohibited comparison of the histology of the two sides. Thus, these two animals were excluded from statistical analysis. The degree of neointimal hyperplasia at each anastomosis was evaluated by both visual scoring (range 0–5) and planimetry in the remaining seven animals. The median hyperplasia score of the treated graft-venous anastomoses was 3.0 (2.05–3.59), whereas the score of the contralateral controls was 2.3 (1.85–2.45; N ¼ 7; P ¼ 0.08; Figure 2a). The median hyperplasia surface area of the treated graft-venous anastomoses quantified by planimetry, normalized to the cross-sectional surface area of the graft, was 0.45 (0.30–0.86). For the controls, it was 0.24 (0.21–0.30; N ¼ 7; P ¼ 0.08 (Figure 2b)). The median hyperplasia visual score of the treated graftarterial anastomoses was 1.40 (0.88–2.30), whereas the median score of the contralateral controls was 1.65 (1.25–2.05; N ¼ 7; P ¼ 0.64; Figure 3a). The median normalized hyperplasia area of the treated graft-arterial anastomoses was 0.12 (0.07–0.39), whereas the median hyperplasia area 2180

a Lumen Lumen

NH Graft

NH Graft

b

c

Lumen Graft

NH

Lumen

NH

Graft

Figure 1 | Histologic appearance of neointimal hyperplasia at the graft-venous anastomosis at different time points. The tissue cross-sections were obtained as described in Figure 7 (cross-section D) and stained with H&E and examined at original magnification, 5. Neointimal hyperplasia (NH) was minimal at the graft-venous anastomosis at (a) 14 days and progressive at (b) 21 and (c) 28 days post-graft placement. Kidney International (2006) 69, 2179–2185

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b 2.0

5

Normalized hyperplasia area

Hyperplasia score

a

4 3 2 1

1.5 1.0 0.5 0.0

0 Control

Dipyridamole

Control

Dipyridamole

Figure 2 | Effect of local delivery of dipyridamole on neointimal hyperplasia at graft-venous anastomoses. Dipyridamole was delivered using microspheres/ReGel or ReGel alone as a perivascular sustained-release depot around the graft-venous and graft-arterial anastomoses as described in Table 1. The degree of neointimal hyperplasia was assessed by (a) visual scoring (scale of 0–5, with 5 representing maximal hyperplasia) and (b) by planimetry. The planimetry results are presented as the ratio of the cross-sectional surface area of the hyperplastic tissue, normalized to the crosssectional surface area of the graft, resulting in a dimensionless value. The thin lines connecting the control and dipyridamole-treated values represent individual animals, whereas the thick line represents the median value of all animals. There were no statistically significant differences between the mean control values and the dipyridamole-treated values as assessed by either scoring (N ¼ 7; P ¼ 0.08) or planimetry (N ¼ 7; P ¼ 0.08).

b

5

Normalized hyperplasia area

Hyperplasia score

a

4 3 2 1

2.0 1.5 1.0 0.5 0.0

0 Control

Dipyridamole

Control

Dipyridamole

Figure 3 | Effect of local delivery of dipyridamole on neointimal hyperplasia at graft-arterial anastomoses. The methods of drug administration and assessment of hyperplasia are similar to those described in Figure 2. (a) Results of visual scoring. (b) Results by planimetry. There were no statistically significant differences between the median control values and the dipyridamole-treated values at the graft-arterial anastomoses as assessed by either scoring (N ¼ 7; P ¼ 0.64) or planimetry (N ¼ 7; P ¼ 0.31).

Systemic effects of locally delivered dipyridamole

There were no noticeable physical problems, such as generalized or localized bleeding, in the treated animals. All complete blood counts and blood chemistries were within the normal range. Peripheral plasma dipyridamole concentrations were consistently below the detection limit of 10 ng/ml at all time points. DISCUSSION

Because of the chronic nature of the stimuli, long-term preservation of dialysis PTFE grafts likely requires the sustained delivery of antiproliferative agents. Although daily Kidney International (2006) 69, 2179–2185

Figure 4 | Histologic examination of a drug depot at the graftvein anastomosis. Presented is the histologic cross-section stained with H&E at the graft-venous anastomosis of a graft that received dipyridamole in microspheres/ReGel intraoperatively and explanted after 28 days. The original magnification of the main figure is 1. The drug depot was encapsulated with fibrous tissues, which was confirmed using trichrome staining (not shown). The inset (original magnification, 20) is an enlargement of a segment of the depot, showing fibrous tissues surrounding the microspheres, infiltrated by mononuclear cells and giant cells (black arrow).

oral administration is a possibility, local delivery of agents targeting the likely foci of hyperplasia development allows far higher local drug concentrations while minimizing systemic side effects. There are a variety of techniques for local drug delivery, including the coating of the graft or intraluminal stent with antiproliferative agents. The latter approach requires an invasive procedure and the lodging of an additional foreign object, with the associated risks of further endothelial injury, thrombosis, and infection. Neither technique permits the replenishment of the antiproliferative agent. In contrast, the percutaneous injection of a sustaineddelivery platform would allow for the repeated replenishment of the drug depot at the superficial perivascular locations. The thermosensitive triblock polymer ReGel appears to fulfill these requirements.11 Indeed, our recent study suggests that the delivery of paclitaxel using this system was effective in inhibiting neointimal hyperplasia in a canine model.9 Although ReGel can solubilize and stabilize a variety of substances, hydrophilic drugs such as dipyridamole are usually retained for shorter durations in the gel than those with greater hydrophobicity. The incorporation of dipyridamole into polymeric microspheres before mixing with ReGel substantially decreased the initial burst and extended its release.12 This combination was therefore used to provide injectable, sustained delivery of dipyridamole in our porcine model. A large number of substances have been reported to possess antiproliferative properties against arterial smooth muscle cells, although their efficacies against venous smooth muscle cells have not been extensively tested.10 If effective, the use of small-molecule drugs is probably simpler, safer, and less expensive than biological reagents, such as antibodies and oligodeoxynucleotides. Our selection of dipyridamole in 2181

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b 1000

1000

Dipyridamole (g/ml)

Dipyridamole (g/ml)

a


100

10

100

10

1

1 0

7

21 14 Time (days)

28

0

7

14 21 Time (days)

28

Figure 5 | Time-dependent changes in dipyridamole concentrations in venous and arterial walls at graft anastomoses. Dipyridamole (52 mg) was delivered using microspheres/ReGel as a perivascular sustained-release depot around the graft-venous and graft-arterial anastomoses. The graft and adjoining native vessels were explanted at the indicated time points after graft placement. Dipyridamole was extracted from the venous wall (a) and the arterial wall (b) and assayed. N ¼ 3 at 0.5, 1.5, 4, and 8 days and N ¼ 2 at 21 days.

testing the ReGel system is based on previous in vitro and in vivo results. In vitro, dipyridamole markedly inhibits the proliferation of both human arterial and venous smooth muscle cells,8,13 with potency greater than that of paclitaxel.10 We also found that dipyridamole inhibited the proliferation of porcine arterial and venous smooth muscle cells, with potencies similar to those in human vascular smooth muscle cells (unpublished data). In addition, venous smooth muscle cells were more sensitive to dipyridamole than arterial cells, for both human10 and porcine cells. The IC50 values (concentrations required to inhibit cell proliferation by 50%) were 12.771.6 and 16.671.0 mg/ml for porcine venous and arterial cells, respectively (unpublished data). This observation is significant as neointimal hyperplasia occurs more commonly at the venous anastomosis than the arterial anastomosis of the arteriovenous graft. Not only does dipyridamole inhibit the proliferation of normal human and porcine cells,8,13 but is also effective in vitro against vascular smooth muscle cells obtained from the hyperplastic lesions in our porcine model (unpublished data). In a rabbit model, the continuous infusion of dipyridamole into the adventitia for more than 2 weeks partially inhibited intimal thickening after balloon injury of the carotid artery, providing further impetus to test this apparently promising drug in a perivascular delivery model.8 It should also be further noted that dipyridamole provides certain technical advantages for in vivo pharmacokinetic analysis of a new drug delivery system in that it is fluorescent and can be readily detected in blood and tissue samples. Despite the proper localization of ReGel around the anastomoses (Figure 4), the perivascular delivery of dipyridamole using this system failed to prevent neointimal hyperplasia. In fact, there was a trend towards more severe hyperplasia at the graft-venous anastomosis associated with dipyridamole treatment, compared to untreated controls (Figure 2). These results were consistent using either the 2182

visual scoring or planimetry technique for analysis. The explanations for this lack of efficacy are not apparent. Our pharmacokinetic results in vitro and ex vivo have shown that dipyridamole readily diffuses into and through the wall of the PTFE graft, native porcine artery, and native porcine vein.14 The present study also demonstrated the presence of significant concentrations of dipyridamole in the venous and arterial walls around the anastomoses (Figure 5). These tissue concentrations were 10- to 20-fold higher than the in vitro IC50 values for porcine vascular smooth muscle cells and decreased to IC50 values by 3 weeks. Thus, these pharmacokinetic data suggest that the antiproliferative effects of dipyridamole should have been exerted on the neointima for most of the study period. One potential explanation for the lack of efficacy in the animal model is that the cell culture conditions used to characterize antiproliferative effects of dipyridamole do not properly mimic conditions in vivo, which is always a consideration in extending in vitro results to in vivo applications. For instance, neointimal cells in vivo may be exposed to high concentrations of nitric oxide produced by inflammatory cells or damaged endothelium. This might result in the activation of guanylyl cyclase to produce high, sustained intracellular concentrations of cGMP in neointimal cells, comparable to concentrations achieved in vitro as a result of phosphodiesterase inhibition by dipyridamole. It is also possible that neointimal cells in vivo are exposed to a milieu of mitogenic stimuli that are very different from that encountered by cells in vitro and that the activation of these different mitogenic signaling pathways is largely insensitive to the antiproliferative effects of dipyridamole. Local delivery of dipyridamole using microspheres and ReGel appeared to be safe, even up to 104 mg per animal. No systemic bleeding or other adverse effects were noted. This is perhaps not surprising considering the slow release of the total amount of drug over 3 weeks, in conjunction with the hepatic metabolism of the drug. These resulted in no detectable drug level in the peripheral blood throughout the study period, in contrast to the usual clinical dose of 300 mg/day orally, which results in much higher systemic concentrations. There were also no adverse events, such as bleeding or delayed healing around the surgical wound. There was, however, an apparent foreign body reaction to the drug depot in four of the nine animals (44%), characterized by the presence of mononuclear cell and giant cell infiltration as well as fibrous capsule formation. Although it is difficult to ascertain the exact culprit responsible for this reaction, the administration of ReGel into rodents11 and dogs9 has not produced significant local inflammatory responses. In contrast, the administration of poly(lactide-co-glycolide) microspheres with diameters o10 mm15 subcutaneously into rats has been reported to produce similar reactions. We also found similar reactions histologically in pigs after the perivascular administration of ReGel mixed with microspheres and without dipyridamole (unpublished data). Thus, it is highly likely that these foreign body reactions observed in Kidney International (2006) 69, 2179–2185


our present experiments were due to the presence of microspheres. It should, however, be noted that this reaction was seen in only a subset of animals tested. It is also likely that the employment of fewer microspheres of larger sizes may markedly diminish this reaction.15 There are limitations to the present study. First, aspirin and clopidogrel were employed to prevent thrombosis in order to study the development of neointimal hyperplasia, similar to the porcine PTFE graft models reported by other investigators.16–20 In addition to its anti-platelet effects, aspirin has also been shown to potentiate the proliferation of human vascular smooth muscle cells induced by plateletderived growth factor,21 which might have confounded the results in the present study. Both control and experimental grafts, however, were exposed to this drug, which was administered systemically. Second, the animals in this and other porcine models16–20 were normal rather than uremic. As such, the pathophysiology of the hyperplasia and the response to pharmacological treatment may potentially be different from uremic animals. Although this study showed the general utility of the microsphere/ReGel system as a sustained local drug delivery system, it is unlikely that dipyridamole will be effective for the prevention of neointimal hyperplasia associated with hemodialysis arteriovenous PTFE graft stenosis when delivered in this manner. Other antiproliferative drugs with different mechanisms of action, particularly those that are more hydrophobic and thus can achieve longer sustained release from ReGel, should be investigated. MATERIALS AND METHODS Preparation of dipyridamole-incorporated microspheres ReGel (MacroMed Inc., Sandy, UT, USA) is a polymeric gel designed for the sustained release of drugs. Because of its thermosensitive nature, liquid ReGel can be mixed with drugs in vitro and injected into the body, where it rapidly transforms into a semi-solid gel and provides a sustained-release drug depot.11 Although ReGel can solubilize and stabilize a large variety of drugs, the low hydrophobicity of dipyridamole may diminish the sustained-release kinetics from the gel. In order to prolong the release, dipyridamole was first incorporated into poly(lactide-co-glycolide) (Medisorbs, lactide:glycolide 50:50 (v/v), M n 28 500 Da, Alkermes Inc., Cambridge, MA, USA) microspheres using an oil-in-water emulsion method.12 The microspheres that were prepared in this manner were approximately 2–10 mm in diameter. Considering that the initial drug loading level was 30% (i.e., 30 mg dipyridamole per 70 mg poly(lactide-co-glycolide)) and the encapsulation efficiency of dipyridamole in the microspheres was 55.0%, the final preparation used in animal experiments contained 52 mg of dipyridamole per 315 mg of microspheres. Preparation of mixture of dipyridamole or dipyridamole/ microspheres with ReGel Dipyridamole/microspheres were sterilized by gamma irradiation (Isomedix Inc., Whippany, NJ, USA) using 60Co as irradiation source at a dose of 15 kGy. Dipyridamole (26–52 mg) in microspheres or dipyridamole (0.26–26 mg) alone was mixed with 2 ml of ReGel and maintained at 41C until use. Kidney International (2006) 69, 2179–2185

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Porcine graft model A total of 29 pigs were used: 11 for establishing the model, nine for examination of drug efficacy, and nine for determination of tissue drug concentrations. The porcine model of PTFE graft was established as follows. All animal procedures and care were performed in accordance with the ‘Principles of Laboratory Animal Care’ and the ‘Guidelines for the Care and Use of Laboratory Animals’ (NIH Publication No. 85-23, revised 2001) and were approved by the Institutional Animal Care and Use Committee of the University of Utah and the Veterans Affairs Salt Lake City Healthcare System. Yorkshire cross domestic swine, weighing 30.970.9 kg, were used. General anesthesia was induced using xylazine (4 mg/kg), telazol (4 mg/kg), and ketamine (4 mg/kg). The trachea was then intubated and general anesthesia was maintained using 1.5–2.5% isoflurane. Sodium heparin (100 U/kg) was given as a single intravenous bolus at the beginning of the surgery. Under sterile conditions, expanded PTFE grafts (spiral re-enforced, 7-cm length, 6-mm internal diameter; Bard Peripheral Vascular Inc., Tempe, AZ, USA) were placed between the common carotid artery and ipsilateral external jugular vein bilaterally. As this study specifically targeted neointimal hyperplasia, adjunctive anti-platelet agents were used to minimize the risk of thrombosis. Starting 6 days preoperatively, aspirin EC (Phamaceutical Formulations, Edison, NJ, USA) at 81 mg/day was administrated orally. Clopidogrel (Bristol-Myers Squibb, New York, NY, USA) at 225 mg was added 1 day before surgery and continued at 75 mg/day postoperatively. Both anti-platelet agents were continued until death occurred. Enrofloxacin (Baytril, Bayer, Pittsburgh, PA, USA) was administrated subcutaneously at 5 mg/kg/day starting on the day of surgery and was continued for 3 days. For ultrasound, blood sampling, and postoperative injection of dipyridamole, the animals were sedated using intramuscular ketamine, xylazine, and telazol, each at 4 mg/kg. Dipyridamole administration In four animals, immediately after graft placement, a mixture of dipyridamole (26–52 mg)/microspheres and ReGel (2 ml) was applied intraoperatively to the external surface of each of the graft-venous and graft-arterial anastomoses on one side of the animal (Figure 6). In order to ensure that the drug depot was held in place at the anastomoses before tissue in-growth, the surrounding tissues were sutured before wound closure. The contralateral graft received no treatment and served as control. In five other animals, 1 week after graft placement, dipyridamole (0.26–26 mg) directly mixed with ReGel (2 ml) was injected into the perivascular area around each anastomosis using an 18-gauge needle and a 3-ml syringe under ultrasound guidance (see Table 1) on one side of the animal. The contralateral graft received no treatment and served as control. In all cases, the side that received the drug was selected at random in a blinded manner. See Table 1 for summary of dipyridamole administration. Graft patency monitoring and blood sampling Patency of grafts was monitored weekly using a Doppler ultrasound (SonoSite, Bothell, WA, USA) and an L38/10-5 MHz transducer (TITAN, Sonosite). Peripheral blood was obtained from an ear vein weekly for complete blood count, chemistry, and dipyridamole concentration. Tissue processing for histology At various time points after graft placement, the animals were killed by an intravenous injection of pentobarbital (100 mg/kg) and the 2183

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grafts were explanted en bloc along with a 2-cm-long segment of the adjacent blood vessels. The explanted grafts were immediately fixed in 10% buffered formalin and embedded in paraffin. Two standardized cross-sections were cut at each anastomosis: one at 3 mm from the proximal edge and the other at 3 mm from the distal edge (Figure 7). The slides were stained with H&E and trichrome as indicated. Assessment of neointimal hyperplasia by scoring Each slide of the anastomotic cross-sections was evaluated for neointimal hyperplasia in the graft using two different methods. The first was standard visual scoring by four independent observers who were blinded to the treatment that the particular graft received.9 The scoring ranged from 0 to 5, with 0.5-unit increments. Any tissue ingrowth overlying the graft lumen was considered to be neointimal hyperplasia, with minimal lining of the graft being 0.5 and total occlusion of the lumen by hyperplasia arbitrarily scored as 5. An exemplary slide was used to define the score of 0.5 before scoring started.

Common carotid artery

PTFE graft

Spiral enforcement

External jugular vein

Cephalad

Caudal

Figure 6 | Photograph of ReGel mixed with dipyridamoleincorporated microspheres deposited at PTFE graft anastomoses. An arteriovenous PTFE graft was created between the common carotid artery and the external jugular vein. Liquid ReGel mixed with dipyridamole-incorporated microspheres was applied to the graft-arterial and the graft-veinous anastomoses (m). This liquid transformed rapidly into a solid gel to provide a drug depot. The yellow color of the gel was imparted by dipyridamole.

Quantification of neointimal hyperplasia by planimetry Digital images of the H&E-stained cross-sections of each graft-vessel anastomosis were obtained at original magnification, 1 using a CCD camera connected to a dissecting scope. The outline of the hyperplastic tissue within the graft lumen on the computerized images was determined by an independent blinded reviewer and the surface area quantified using BioQuant Software (BioQuant Image Analysis Corporation, Nashville, TN, USA). Determination of tissue dipyridamole concentrations In four pigs, a mixture of dipyridamole (52 mg)/microspheres and ReGel (2 ml) was applied intraoperatively to the external surface of each of the graft-venous and graft-arterial anastomoses on one side of the animal. The contralateral (control) side received no treatment. The animals were killed at 0.5, 1.5, 4, or 8 days after graft implantation, respectively. As no dipyridamole (o10 ng/g) could be detected in the vessel walls on the control side (i.e., there was no transport of the drug from the other side), subsequent experiments were performed in five additional pigs in which bilateral grafts received a mixture of dipyridamole (52 mg)/microspheres and ReGel (2 ml) at each anastomosis. These five animals were killed at 0.5, 1.5, 4, 8, or 21 days after graft implantation, respectively. The graft along with the tissues between the two anastomoses was explanted en bloc. The anastomoses were freed from surrounding tissues by careful dissection. A 1-cm-long segment of the native artery or vein immediately adjacent to the drug depot was isolated and weighed. Dipyridamole was extracted from each section by immersion into 1 ml of 100% ethanol. This vessel–ethanol mixture was homogenized for 2 min on ice using a homogenizer (PowerGen 700, Fischer Scientific, Pittsburg, PA, USA) and centrifuged at 1500 r.p.m. for 10 min. The dipyridamole in the supernatant was assayed using a spectrofluorometer at an excitation wavelength of 412 nm and an emission wavelength of 495 nm.22 The tissue drug amount was converted to concentrations assuming the tissue density to be 1 g/cm3. Determination of plasma dipyridamole concentrations Systemic exposure to locally delivered dipyridamole was assessed by determination of dipyridamole concentrations in peripheral blood. Blood samples were obtained weekly from an ear vein. Plasma concentrations of dipyridamole were measured as described above.

Table 1 | Dosage, time, and system for the local delivery of dipyridamole for individual animals Animal no.

Time of dipyridamole application

Delivery system

1 2 3 4 5 6 7 8 9

1 week 1 week 1 week 1 week 1 week Intraoperative Intraoperative Intraoperative Intraoperative

ReGel ReGel ReGel ReGel ReGel ReGel+Msph ReGel+Msph ReGel+Msph ReGel+Msph

Dose of dipyridamole per anastomosis (mg)

Time of killing (weeks)

0.26 0.26 2.6 2.6 26 26 52 52 52

3 3 3 3 3 3 4 4 4

The specified dosage of dypyridamole was mixed with 2 ml of ReGel and injected in liquid form under ultrasound guidance to the perivascular area surrounding each of the venous and arterial anastomoses of one graft at 1 week (animal nos. 1–5) or incorporated into microspheres (Msph), mixed with 2 ml of ReGel and applied in gel form to the perivascular surface of each of the venous and arterial anastomoses of one graft at the time of graft placement (animal nos. 6–9). The contralateral grafts received no treatment and served as controls. Animal nos. 2 and 3 were excluded from statistical analysis of the hyperplasia because of technical problems. Bilateral grafts were patent at the time of killing in all the remaining animals.

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PTFE graft

Cross-section

NH

5.

6.

7.

DP

8. Native vessel

Figure 7 | Cross-sections of the graft-vessel anastomoses for histologic analysis. Each graft-arterial or graft-veinous anastomosis was cut along the two dotted lines to produce cross-sections for histologic analysis of neointimal hyperplasia. The cross-section designated ‘P’ was standardized at 3 mm from the proximal edge, whereas the cross-section designated ‘D’ was standardized at 3 mm from the distal edge of the anastomosis. NH, neointimal hyperplasia.

9.

10.

11.

Statistical analysis Results of planimetry and visual scoring are expressed as the median and the 25th and 75th percentile range. The Wilcoxon signed rank test was used to compare the two groups. P-values o0.05 were considered statistically significant.

12.

ACKNOWLEDGMENTS

15.

This work was supported by the National Heart, Lung and Blood Institute (RO1HL67646), Medical and Research Services of the Department of Veterans Affairs, Dialysis Research Foundation, and National Kidney Foundation of Utah & Idaho. Expanded PTFE (Impras) grafts were kind gifts of Bard Peripheral Vascular Inc. We thank Ilya Zhuplatov for technical assistance in animal studies and Sreevalli Sikharam in planimetry. REFERENCES 1. Kanterman RY, Vesely TM, Pilgram TK et al. Dialysis access grafts: anatomic location of venous stenosis and results of angioplasty. Radiology 1995; 195: 135–139. 2. Swedberg SH, Brown BG, Sigley R et al. Intimal fibromuscular hyperplasia at the venous anastomosis of PTFE grafts in hemodialysis patients. Clinical, immunocytochemical, light and electron microscopic assessment. Circulation 1989; 80: 1726–1736. 3. Maurice DH, Palmer D, Tilley DG et al. Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system. Mol Pharmacol 2003; 64: 533–546. 4. Sreedhara R, Himmelfarb J, Lazarus JM et al. Anti-platelet therapy in graft thrombosis: results of a prospective, randomized, double-blind study. Kidney Int 1994; 45: 1477–1483.

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